PYMOL TUTORIAL by techmaster

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									                 PYMOL TUTORIAL

           PART OF BIOCHEMISTRY 712 AND BIOCHEMISTRY 660
                     UNIVERSITY OF WISCONSIN-MADISON




MAJOR PROFESSOR:
                                Ann Palmenberg
           Institute for Molecular Virology & Department of Biochemistry
                                 acpalmen@wisc.edu

THIS TUTORIAL WRITTEN BY:
                               Dr. Jean-Yves Sgro
   Biology New Media & Biotechnology Centers, & Institute for Molecular Virology
                                jsgro@wisc.edu


QUESTIONS/ANSWSERS SECTION THANKS TO

                                 Adam Steinberg
                       Department of Biochemistry Medialab
                              hsteinbe@wisc.edu


                                                                     version 11/2006
BIOCHEMISTRY 660 / 712 – FALL 2006                                                  J.Y. SGRO




             L02: DESKTOP MOLECULAR GRAPHICS III: PyMol
                                          CONTENTS

PyMol - Exercise A: Download a PDB from the repository
PyMol - Exercise B: Open PyMol and load a PDB file
PyMol - Exercise C: PyMol interface
PyMol - Exercise D: Action preset menus
PyMol - Exercise E: Useful commands to analyze structure and create images
PyMol - Exercise F: A simple animation within PyMol, and for PowerPoint
PyMol - Exercise G: Harnessing the power of PyMol: introducing scripts
PyMol - Exercise H: Select command, parameters, scripting, and subsets.

                         =========================================

From the preface of the User’s guide: “PyMOL was created in an efficient but highly pragmatic
manner, with heavy emphasis on delivering powerful features to end users. Expediency has
almost always taken precedence over elegance, and adherence to established software
development practices is inconsistent. PyMOL is about getting the job done now, as fast as
possible, by whatever means were available.”
PyMol is a multiplatform molecular graphics software with many advanced features such as
rendered cartoon ribbons and surfaces, internal ray tracing and movie tools and is fast
becoming the new “standard” in molecular graphics. The PyMol web site is located at
http://pymol.sourceforge.net/

                         =========================================

Note: within the exercises, Bold text shows what actions are taken by the user : typing text or
clicking the mouse.

                         =========================================

PyMol - Exercise A: Download a PDB from the repository

Reminder: Structures have a PDB ID code made of 4 letters and numbers. PDB files contain
coordinates pertinent to the crystallographic arrangement of the molecules within the crystal.
The biological functional entity can be either a multimer of the deposited structure, or just one
of multiple copies within the file. In the following example we will download one functional
biological subunit, in this case a monomer.

    1) Open a web browser such as Safari or Firefox.
    2) Point your web browser to www.rcsb.org
    3) In the Search box enter the following ID: 2biw and click SEARCH button




                                           L02 – page 1
BIOCHEMISTRY 660 / 712 – FALL 2006                                                 J.Y. SGRO




    4) On the left column click Download
       Files to show submenus
    5) Click the first option Biological Unit
       Coordinates 2

    Note: DO NOT USE Biological Unit
    Coordinates 1

    6) The file is saved on the desktop as
       2BIW.pdb2




You can now close your browser, or hide it (Command-H).

                         =========================================

PyMol - Exercise B: Open PyMol and load a PDB file

                      PyMol or MacPyMol should be located within the Applications > Classes at
                      the BNMC computer. Your instructor may give you a different location if
                      necessary.



    1) double-click on the PyMol icon to launch the
       software.

Note: Some versions of PyMol have the top and bottom
panels in separate windows, but offer the same interface.




    2) Type the next commands after PyMOL> within the top line command:




PyMOL> cd desktop                             Note the echo on the text area above
PyMOL> pwd                                   this will echo /Users/BNMC/Desktop or a similar
                                             path. On a Windows system the path would begin
                                             with C:\
PyMOL> load 2BIW.pdb2                        PyMOL>load 2BIW.pdb2
                                             HEADER    OXIDOREDUCTASE                    19-JAN-
                                             05   XXXX

                                             L02 – page 2
BIOCHEMISTRY 660 / 712 – FALL 2006                                                         J.Y. SGRO


This will load the structure and echo          TITLE     CRYSTAL STRUCTURE OF APOCAROTENOID CLEAVAGE
                                               OXYGENASE FROM
information in the text panel as               TITLE    2 SYNECHOCYSTIS, NATIVE ENZYME
                                               COMPND    APOCAROTENOID-CLEAVING OXYGENASE
reproduced to the right.                        ObjectMolecule: Read secondary structure assignments.
                                                ObjectMolecule: Read crystal symmetry information.
                                                Symmetry: Found 4 symmetry operators.
Note: file name is case sensitive               CmdLoad: "2BIW.pdb2" loaded as"2BIW.pdb2".



Note: why not use the File> Open menu sequence? Good question…
You can actually try that option and it might work on your current
system. However you will likely find that when you get to the Open
form the files you want to open are listed in gray and therefore cannot
be opened. The easy fix is to simply rename the file from *.pdb1 or
*.pdb2 to simply *.pdb before you try to open it.


                         =========================================

PyMol - Exercise C: PyMol interface


This exercise continues on the
previous exercise where 2BIW.pdb2
was loaded within the PyMol
software.

Your screen should be similar to this
image without the extra markings.

The Viewer and Internal GUI are the
parts we will use most.

The default size of the image in the
viewer is 640 x 480 pixels.




         The menu bar might be attached to the external GUI in systems other than Mac.

By default PyMol will display the molecule(s) contained within the PDB file as a wireframe.

This cyanobacterium molecule is a carotenoid oxygenase and contains a carotenoid ligand.

    1) Mouse control of the 3D representation

The 3D molecule is represented within a virtual 3D world. The flat surface of the screen
represents the X and Y axes while the Z, depth axis is perpendicular to the screen.


                                               L02 – page 3
BIOCHEMISTRY 660 / 712 – FALL 2006                                                         J.Y. SGRO


PyMol is optimized for a 3-button mouse but most basic functions can still be achieved by a
one-button mouse, in particular the rotations around X, Y, and Z.

 Rotation around the X or Y axis:          (left) click and drag.
 Rotation around the Z axis:               (left) click on the top left or right corner.
 Translate (move sideways) X or Y:         click middle button and drag.
 Zoom (move along Z axis):                 click right button and drag up or down.

    2) Changing the default mouse settings

By default PyMol assumes that you have a 3-button mouse. If you have a 2-
or one-button mouse you can change the setting accordingly with the
“Mouse” menu in the top menu bar.
Note the updating of the “Mouse Mode” button mapping at the bottom right
within the “Internal GUI” if you change the mouse setting.


Note: the Selection Mode> submenus define what is
selected when an atom is clicked on. The selection
default is Residues.


    3) Changing the representation of the molecule

PyMol can open more than one molecule at a time, or separate complex PDB files into
individual components. Each opened or loaded molecule is given a name within the “Names
Panel” (see picture above). The first name is always “all.” Clicking on the name itself will
undisplay the corresponding molecule(s) (temporarily invisible).

The ASHLC menu (           ) is abbreviated for Action, Show, Hide Lablel and Color.
Some menu items have submenu components. Selections made under the “all” line will affect
all the opened molecules.


 Rule: Once a selection is shown (S) it must be selectively hidden (H) as it is not removed
 when another selection (S) is made. Selections are therefore additive, which allows for the
 creation of images with mixed graphical representations.

 Let’s first make a cartoon representation of this protein: within the
 2BIW line click S and select Cartoon. The molecule is now shown
 as both cartoon and wireframe. Remove the wireframe by clicking
 H and lines.

 (Note that within the S menu list, the “as” menu contains a lot of
 redundancies.)



                                           L02 – page 4
BIOCHEMISTRY 660 / 712 – FALL 2006                                              J.Y. SGRO


 Options: most options can be set within the “Setting”
 menu within the top menu bar.

 For example, it is possible to change the way all alpha
 helices are rendered.

 Go to the following menu cascade: Setting > Cartoon >
 Cylindrical Helices
 Select this option again to remove its effect and do the
 following: Setting > Cartoon > Fancy Helices

Testing other cartoon settings: Engaging the option Smooth Loops will simplify the drawing.
Removing the option Highlight Color will make the edges of strands and inside helices
surfaces a gray color (default).

Black backgrounds look very nice on the screen but do not print well on paper and do not
photocopy well. Changing the backgroud to white is usually very useful:

 The “Display” menu within the top menu bar contains options for
 most options pertinent to displaying the image witin the PyMol
 viewer.

 To change the background color to white follow this menu
 cascade:
 Display > Background > White

 Note that there is “fog” within the back of the molecule, which can
 be toggled on and off with the Depth Cue menu item within this
 same menu list.



 Changing the color of the ribbon is easy with the following cascade
 menu within the PyMol “Names Panel” of the “Internal GUI” under the C
 menu as shown in the following menu cascade:

 2BIW.pdb2 > C > oranges > orange

 You may also choose another option which is to color by secondary
 structure by following this menu cascade instead:

 2BIW.pdb2 > C > by ss > Helix Sheet Loop (choose one of the the 2
 color options displayed e.g. pink and cyan.




    4) changing the representation of the molecule: adding ligand
                                           L02 – page 5
BIOCHEMISTRY 660 / 712 – FALL 2006                                                          J.Y. SGRO




When we opted to show the molecule as a cartoon above, one thing happened: the protein
was shown as the familiar cartoon represenation, but if any ligand is present (which is the true
for this file) it simply disappears as it is not part of the cartoon representation of the protein.
Here we will “rescue” the ligand!

 This menu cascade is also within the “ASHLC” menus of the PyMol
 “Internal GUI” “Names Panel.” Since ligands are usually small, organic
 molecules, the following cascade within the Show menu for line 2BIW
 will show the ligand. Perform the following cascade:

 2BIW.pdb2 > S > organic > spheres

 This cascade will select the carotenoid present witin the PDB file.
 Note that it is the same color as other parts of the protein, as it is part of the name
 2BIW.pdb2 line. It will be either orange or the color for loops in the C/ss/Helix-Sheet-
 Loop color scheme chosen in the step above.




    5) Mouse selection

Now that we have made the ligand visible, it becomes easier to select it with the mouse to
make further changes.

Click on one of the spheres of the carotenoid ligand. This simple click makes various things
happen:

           a. pink, square dots appear onto all the spheres of the lignad, indicating that it has
               been selected.
           b. A new name line appears within the “Names Panel” GUI called “(sele)” and is
               now dedicated to this subset of atoms. Note that the content of (sele) changes as
               other atoms are clicked
           c. The name of the atom that was clicked appears within the top text window of the
               “external GUI.” For example:
                      You clicked /2BIW.pdb2//B/TYR`322/OH
                      Selector: selection "sele" defined with 12 atoms.
        This could be read as “you clicked atom OH which is on the 322nd atom in the file, and
        belongs to Tyrosine 322 of chain B in the object created when opening file 2BIW.pdb2.
        The complete selection contains 12 atoms (which make up the complete Tyrosine
        without the hydrogens, since these are usually not present in PDB files.)

Now that the ligand atoms are segregated within the name of (sele) we can change its color if
we want by the following cascade within the Names Panel:

(sele) > by element > CHNOS….
(selecting the first CHNOS… after HNOS… in the list would display the ligand as green with
one red oxygen).


                                                     L02 – page 6
BIOCHEMISTRY 660 / 712 – FALL 2006                                                           J.Y. SGRO


    6) Final image(s)

If you still see the selection dots over the ligand from the previous section simply click
anywhere on the white background to unselect. Alternatively click on the “Hide-Sele” button at
the top right hand side of the “external GUI.”

Rotate the molecule to find a perspective that you deem instructive of the conformation of the
protein and it’s bound ligand.

 Follow this menu cascade to save the image currently
 within the Viewer:

 File > Save Image…

 Then replace the default word “pymol” to give a name to
 the file you want to save, e.g. image1
 The image will be saved as a PNG image on the desktop




However this image is rather crude in terms of graphics and resolution. PyMol offers an
internal “ray tracer” to create stunning rendered images with a high visual quality much more
pleasant to the eye and ideal for publication.

 To create a standard ray-traced image of the current Viewer
 scene, click the “Ray” button at the top right of the “external
 GUI.” This will take a few seconds to a few minutes depending on
 the complexity of the PDB file and the the chosen display, and
 will also depend on the speed of the computer CPU. Once
 rendered, the image appears within the Viewer. To save the file,
 use the save cascade as above: File > Save Image…




      Zoomed side-by-side comparison between the pymol image and the ray-traced image: note the jagginess
      of the original image and the smooth appearance of the ray-traced image, with shadows as a bonus.



                                                L02 – page 7
BIOCHEMISTRY 660 / 712 – FALL 2006                                                   J.Y. SGRO


This is useful to create preview images for slide presentations, but is still one step short of the
quality needed for high resolution publication, as the image created by the default setting is the
same size as that of the current Viewer (default size is 640 x 480 pixels when PyMol is first
opened.) The default file format is the new PNG standard. All we need to do at this point is
change the default setting of the ray-traced file size.

 Within the PyMOL> line command type
 the following command to change the
 dimensions of the final ray-traced
 image:

  ray 2000, 2000

 (2000 is an example number. Note the
 comma between the two numbers. The
 higher the numbers the longer it will
 take to compute the image). PyMol will
 display the size of the calculated image
 within the Viewer under the image here
 2000 x 2000

 Note: ray 2000 is also a valid
 command. In this case PyMol will
 evaluate the missing value and adjust it
 to minimize the file size.


                         =========================================


PyMol - Exercise D: Action preset menus

This is mostly a self-paced exploration of one of the menus that is sure to change over time as
PyMol evolves.

If you are not continuing this exercise from the previous exercise load the structure
2BIW.pdb2 as in Exercise B, then click S> Cartoon to be in a similar sate as in the previous
exercise.

    1) The preset menu: from default to more complex

The preset menu is part of the Action set of the ASHLC menus controlling the aspect of
molecules from the Names Panel.

Follow this menu cascade sequence to return to the default view, as when you just opened the
molecule. However no rotation occurs:


                                            L02 – page 8
BIOCHEMISTRY 660 / 712 – FALL 2006                                              J.Y. SGRO


 2BIW.pdb2 > A > preset > default

 This command has a similar effect but is not the
 same as the following cascade: hide everything and
 show lines:

 2BIW.pdb2 > H > everything
 and
 2BIW.pdb2 > S > lines

 Note: the “preset” options will set some variables that
 are specific to these views and may change further
 drawings. To remove the effect of these presets
 affecting an object representation, use the
 A>preset>default menu cascade reset parameters.

Note: to get back to the original opening view simply type reset at the PyMOL> line command.

    2) Exploring more

Explore the other menus of this series.
The cascade menu 2BIW.pdb2 > A > preset is assumed in the following commands:


simple

The tracing can then be made thicker by unselecting the
smooth option with the following menu cascade:
Setting > Ribbon > Smooth
Note: a set of 3 histidines is also shown.

                                                              This is not very useful for a
ball and stick                                                large protein such as this.

b factor putty

The segments with the highest temperature factor are shown
as thicker cylinders. Regions of better resolution have thinner
diameter and are usually found at the core of the protein.
Mostly loops in the outside of the protein wobble: the core
portions of the proteins usually appear more stable than the
external loops. This is mostly useful for crystallographers but
is a cool representation.
                        Color domains in separate rainbow colors and shows backbone and
                        side chains.
technical               Note that a subset name appears in the Names Panel
                        (2BIW.pdb2_pol_co) that control the dashed-line hydrogen bonds.

                                           L02 – page 9
BIOCHEMISTRY 660 / 712 – FALL 2006                                                 J.Y. SGRO




pretty and publication create similar images of a
rainbow color cartoon with a stick ligand. Pretty creates
the default cartoon setting, while publication creates an
image very similar to an image created by Molscript*.
(* Per J. Kraulis 1991: MOLSCRIPT: A Program to Produce Both Detailed
and Schematic Plots of Protein Structures. Journal of Applied
Crystallography. 24: 946-950. http://www.avatar.se/molscript/)




ligands

This option will zoom in on the ligand site and show the
protein as backbone except in the near vicinity of the
ligand where side chains are shown. The ligand is
depicted as a thicker cylinder.

Note: to zoom out, simply click on the A in the ASHLC
menu again.


ligand sites

There are a few options available in this submenu, all
pertinent to looking closely at the ligand in it’s binding
pocket.

You can explore a few of them on your own, once you
are done then select the following option: preset >
ligand sites > solid surface


You should obtain a centered, zoomed view of the ligand shown as a stick model within a
partial molecular surface pocket. The colors are the scheme of previously chosen colors, for
example if you tried the “technical” preset earlier the coloring would likely be like a rainbow.
The following quick changes will make a much nicer image: 2BIW.pdb2 > C > yellows > sand

Now with the mouse click carefully anywhere on the ligand stick to select it. As before you will
have a (sele) name within the Names Panel. You can now change the color with the following
menu cascade: sele > C > blues > slate

Of course you can pick your own colors. Make sure that there is sufficient contrast between the
color of the surface and the color of the ligand.




                                                    L02 – page 10
BIOCHEMISTRY 660 / 712 – FALL 2006                                              J.Y. SGRO




If you have not yet done so, rotate the
molecule to select a nice viewing angle.

If you want to add a more stunning effect
click on the “ray” button as we did before to
complete your publication quality picture.

Note: If you are preparing a figure for a black
and white print publication, it might be
advantageous to use the various gray scales,
black, and white within the “grays” option.

                         =========================================


PyMol - Exercise E: Useful commands to analyze structure and create images

We are still working with the 2BIW.pdb2 file loaded above. Review exercise B if you need to
reload the structure.
This exercise assumes you have just completed creating the above view with the preset menu
cascade: 2BIW.pdb2 > A > preset > ligand sites > solid surface

    1) Sequence viewer

The molecule sequence can be shown at the top of the graphical area. To do this follow the
top menu: Display > Sequence on

The sequence appears just below the PyMOL> line command at the top of the Viewer. The
slide cursor underneath can be used to move the sequence viewed further.




By default the one-letter code is displayed. Since we are
looking at a protein, changing the display to the three-letter
code will be useful within the next step . Do this with the
menu cascade:
Display > Sequence Mode > Residues


Now rotate the molecule to view similar to that shown at
right.

With the mouse, click on the tyrosine residue under the
right hand side, which contribute to the pocket surface.
Change it’s aspect to stick with the following menu
cascade: (sele) > S > sticks

                                           L02 – page 11
BIOCHEMISTRY 660 / 712 – FALL 2006                                               J.Y. SGRO


Note: when you clicked, information about the clicked atom appeared within the text box of the
“external GUI,” e.g.:“You clicked /2BIW.pdb2//B/TYR`322/CG.”
Therefore we know that Tyrosine 322 is the one we chose.

Now slide the sequence cursor to the right to move the sequence displayed until you find
the region of the selected TYR. If you have the one-letter code engaged, it appears under the
number 321. However we know it is 322. If you have engaged the three-letter code as
instructed above TYR 322 is shown highlighted within the sequence line.

Now display the sequence line to see residues PHE 303 and HIS 304. With the mouse click
on residue 303 and residue 304 within the sequence line. Note that they are selected
within the graphical window. The arrows within the following image show where you should
look. As before, transform those side chains to a thicker stick with the menu cascade:
(sele) > S > sticks




Quiz question: We just selected amino acids TYR 322, PHE 303, and HIS 304. Can you
describe with one word one important quality of this binding pocket?
Answer: This binding pocket has the following quality: _____________________________

    2) Measuring distances

Distances are measured between two atoms and are expressed in the same unit as the XYZ
coordinates within the PDB file: Angstroms (1Å = 10–10 m).

As an example we shall measure the distance between two atoms within the carotenoid ligand.

Within the top menu select
Wizard > Measurement
(Note: in older PyMol versions Measurement was called Distance.)
This will create a prompt within the Viewer:
“ Please click on the first atom…”
                                                     L02 – page 12
BIOCHEMISTRY 660 / 712 – FALL 2006                                                  J.Y. SGRO




Within the “Internal GUI” a “Measurement” table also appears. It
can also be used to remove measurement objects after they are
no longer needed.

Click on the first atom on the ligand
Click on the second atom on the ligand

(you can select the same atoms as 2 examples within the image
at right, or select alternate atoms)

The default color of the measurement object is yellow. This can
be easily changed with the familiar ASHLC menu for each
measurement, as a new name is entered within the “Names
Panel” for each distance, e.g. “measure01.”
Change the color to white with the following menu cascade:
measure01 > C > grays > white

Quiz exercise: you can also measure the distance between the OH at the tip of TYR 322 and
the nearest ligand atom (C30). Answer: ___________________________________________


When you are done using the “Measurement” panel on the bottom right click Done.
If you no longer need to display the distance object, click Delete All Measurements.

Alternatively you can use the corresponding “A” menu and select the delete option.


    3) Clipping planes

Clipping planes are imaginary planes in the front and back of the molecule. Parts of the
molecule that are outside the planes are “clipped” and therefore invisible. This is very useful for
complex or large structures.


This image represents the molecule seen side-ways
“inside” the computer monitor.
The 2 black lines represent the yon (far away) and
hither (close) clipping planes. These are parallel to
the flat screen of the computer monitor display. The
gray lines converge toward the user’s eye, who is
looking at the molecule on the computer screen.
                                                                                hither   yon

The BNMC computers are equipped with 3-button mice. To move clipping planes press shift
and the right mouse button simultaneously while dragging up and down. As an exercise, try
to remove some of the molecular surface covering the ligand to create a picture similar to
this:
                                           L02 – page 13
BIOCHEMISTRY 660 / 712 – FALL 2006                                                    J.Y. SGRO




Note: we have seen previously that the top menu “Mouse” contains all the possible options,
with a reminder displayed at the bottom right of the “Internal GUI.” The method to adjust the
clipping plane will depend on the number of buttons on your mouse. Use the “Mouse” menu to
adapt your display to your mouse.

    4) Labels

A label containing the residue type and sequence number (e.g. TYR 322) can be added to a
selected residue with the menu cascade (sele) > L > residues

For publications or slide making, labels could be added with e.g. Adobe (PhotoShop,
Illustrator) or Microsoft (PowerPoint) graphic editing software.

At this point you can explore the L menu, knowing that “clear” will remove the mess that may
occur!

    5) Saving a PyMol session

A session file is a binary file containing all the information and graphical displays currently
within PyMol. It is a way to save the “current state”of the software with all that it contains.
Later, the file can be opened and everything is restored.

            a. Saving the session file

From the top menu follow the menu cascade:

File > Save Session

 In the next window enter a name: MySession
and save it on the Desktop to easily find it again.

The susscessful writing of the file will be reported within the
ext window of the “External GUI” and a file called
Mysession.pse on the desktop (.pse is added automatically).

Now quit PyMol: File > Quit


            b. Restoring the session file
                                            L02 – page 14
BIOCHEMISTRY 660 / 712 – FALL 2006                                                                      J.Y. SGRO




While it is possible to simply double click on the MySession.pse icon, this could create
problems if there are multiple copies of PyMol within your computer and even more so if they
are various versions of the software. It is therefore best to first open the PyMol software copy
you want to utilize.

For this exercise: double click on the Applications > Classes > MacPyMol software to open
it.

Within the PyMol top menu follow the menu cascade: File > Open…..
and then select Mysession.pse from the Desktop.

All the PyMol names and selected items are restored as if you had just made them, even if
days have passed since you saved the file. How convenient!

Note: the file has a binary format. There is no information within the file that is useful for any other purpose. The
file for this exercise should likely be slightly less than 1Mb in size.

                          =========================================

PyMol - Exercise F: A simple animation within PyMol, and for PowerPoint

There are various ways to animate and create movies within PyMol. This short exercise is
meant as a simple option to both create a rotation within the PyMol Viewer, and a simple
option for generating a movie suitable for PowerPoint.
At this point you should have the simple pocket surface with the carotenoid shown as stick as
in the previous exercise.

The command mset defines a movie. Since our current level of visualization only contains
one PDB file, the value of mset will be 1. The command mdo defines an action, here this
action will consist of a rotation about the Y axis.

    1) Automatic rotation within the Viewer

The purpose of this set of command is to create a perpetual rotation within the Viewer around
the Y axis with an increment of 5 degrees.

Within the top PyMOL> command line, type the following commands:

mset 1
mdo 1: turn y,5;
Once the commands have been typed, the “vcr” buttons at
the bottom right of the “Internal GUI” become active. We
only need to use the Play (triangle) and Stop (square)
buttons to activate the animation.                                                    |Stop|Play




                                                     L02 – page 15
BIOCHEMISTRY 660 / 712 – FALL 2006                                                  J.Y. SGRO




click the Play button

watch the rotation happen

click the Stop button when done




Note: the mdo command is followed by a colon (:). However in previous versions of PyMol it
was followed by a comma (,). The ending semi-colon (;) is necessary only if other commands
are required. For example, a rotation both within the Y and X axes of 5 degrees each would
be written as:             mdo 1: turn y,5; turn x,5;

    2) Creation of a movie for PowerPoint

The purpose of this short exercise is the creation of a single movie file suitable to be played
independently or imported within PowerPoint.

The creation of movies within PyMol is different depending on the version and on the operating
system. The MacPyMol version of PyMol contains a built-in function to tap within the
QuickTimePRO engine (even if you do not have the PRO license) and can be used to create
movie files. On other systems, the movie is saved as a series of images (in PNG format) which
then need to be assembled manually with e.g. QuickTimePRO, Adobe Premiere or similar
movie making software, meant to compile a series of images (frames) into a single file movie.
This aspect of movie making as well as the indiosyncracies of PowerPoint and movie
compatibility were reviewed in a previous lab or module.

Since we were using some movie/animation features above, the first command to use is
mclear to remove functions and frames that might have been saved.

Modification of the command mset: since we are going to save the file into a QuickTime
movie, we need to define how many frames the movie will contain. For example, to have 36
frames the command will be written as mset 1 x36

Modification of the mdo command: for each frame we need to specify that we want a rotation
around the (e.g.) Y axis of (e.g) 5 degrees. However this would require to declare each of the
(e.g.) 36 frames and for each frame declare the action we want to take (for example a
rotation). Within PyMol the undocumented command util.mroll(start, finish,
loop-flag) does all of this if we have declared the number of frames with mset. A complete
360 degrees rotation is assumed.


Within the top PyMOL> command line, type the following commands:

mclear
mset 1 x36
util.mroll(1,36,1)
                                           L02 – page 16
BIOCHEMISTRY 660 / 712 – FALL 2006                                                J.Y. SGRO




Now follow this menu cascade:

File > Save Movie As > Quicktime (More Choices)…

(Note: this menu available for native Mac version only, usually called
MacPyMol)


When saving this way, the default encoding (codec) is
“Video” and can work well for this exercise. Other codecs
are also available depending on what is installed on your
system.

Change the default 12 frames per second to 24 from the
pull-down menu.

Keep the quality to medium

Press the Save button

The file should be about 2 Mb in size.

Save the file on the Desktop to find it easily. The default
name is pymol.mov and can be changed here if desired.

Press the Save button

Double click the pymol.mov file and watch it.


Within the Mac finder the movie can also be previewed.
Additionally it tells us the dimensions of the movie:
640x480. (Your movie will have the size of the current
Viewer window.)

Indeed PyMol simply saved the 36 frames with these
dimensions and used QuickTime to assemble them. On
non-Mac computers, simply assemble the individual
frames.
Note: to change the size of the resulting movie file or image files use the following command to
set the size of the Viewer: viewport 320,240 to create images and movies of that size (it is
4 times smaller in surface than a 640x 480 image).

    3) Ray traced movies

The ray tracing option is also available when making movies. The movie making is almost
identical as the previous section, but each image is ray-traced before it is saved. The movie
can then be saved as a series of images or as a movie file from the File menu.

                                                   L02 – page 17
BIOCHEMISTRY 660 / 712 – FALL 2006                                                         J.Y. SGRO


To create a ray-traced movie type the following commands within the top PyMOL>
command line:

viewport 320,240
set ray_trace_frames=1
set cache_frames=0
mclear
mset 1 x36
util.mroll(1,36,1)

            a. if you want to save the individual images use the following command:

mpng MyMovie (this command will create MyMovie.0001.png, MyMovie0002.png etc.)

        The images can then be assembled into a movie by an independent software.

            b. If you want to save the movie with QuickTime follow the same procedure as
               above.

Note however that the progression bar (                         ) will remain active, indicating
that the ray-traced images are still available for other commands (for example the mpng save
command above).

Note: ray-tracing is computationally intensive. The bigger the images (viewport size) the longer
it takes. You can see which frame is being rendered by looking at the bottom right of the
“Internal GUI.”

                         =========================================

PyMol - Exercise G: Harnessing the power of PyMol: introducing scripts

GUI interfaces are nice, but they are slow and cumbersome. Like Rasmol, PyMol also has a
line-command interface that we have already used to type specific commands. These
commands are simply lines of text, and therefore can be placed sequentially within a plain text
file called a script. When the script is invoked from the PyMol line command, the commands on
each line of the script are executed, and the final image is shown within the Viewer.

Note: The Py in PyMol refers to Python, a graphical scripting language, and PyMol can be used as a
PyMol algorithm interpreter. However, this is beyond the scope of these exercises. It is useful to know
this, as you might encounter Python scripts for PyMol. The following exercises are restricted to PyMol-
only commands.

    1) setting up for scripts: review of files, directories and path.

On the hard drive, information is arranged in files and folders, and each operating system has
its own way of organizing and calling on files. As a reminder, files contain information such as
lines of text or binary numbers making up an image or a software algorithm, and are organized
throughout the hard drive within directories, also called folders. When you want to invoke a
                                              L02 – page 18
BIOCHEMISTRY 660 / 712 – FALL 2006                                                       J.Y. SGRO


script, PyMol needs to know where it is! There are two fundamentals ways to make sure PyMol
knows where the script you want to use is located:
          a. Give the full path to the script such as
              /Users/BNMC/Desktop/PyMol/myfirstscript.pml on a Mac OSX or
              Unix/Linux system, or perhaps something like
              D:\Data\My Datafiles\PMscripts\myfirstscript.pml for Windows.
              These long lines give the absolute path to the location of the script, and must be
              present at each invocation.
          b. Set the default working directory to where scripts (and PDB files) are located. We
              did something very similar when we gave the command cd desktop in exercise
              B, so that PyMol would automatically look onto the desktop for the necessary
              file(s). In some respect this is easier because once the path has been set with
              the cd command, the path does not have to be repeated again each time we
              want to run this or another script at the same location.

You should be (or become) familiar to the ways directories and paths are dealt with on your computer,
which are essentially the Unix/Linux/OS X family or the Windows operating systems.

    2) Review for Text-only files

This is rather simple but better repeated here for sake of clarity.

A file containing text that is bold, underlined, or italicized it NOT a plain text file (note: there
are exceptions to this rule in Macintosh world not covered here).

A plain text file:

      a. contains ONLY printable characters that can be typed from the simplest of
          keyboards (ASCII characters): letters, numbers and symbols such as !, @, and $.
      b. is not specific to any particular word processor format and is displayed with the
          default font within any word processor (some will choose courier by default for
          this)
      c. contains a “line break” or “end-of-line” code (specific to the operating system) that
          signifies that the line has terminated “here” and that a new line will begin “next.”
Options to create text-only files:

       a. Microsoft Word: any file can be “Saved As…”
However MS Word is not a good choice to edit text-only files as the Save
As… may need to be repeated after each modification of contents.
Note: the “Line Breaks” refer to the way lines currently wraps around in the
displayed text. For scripts Text Only should be preferred.

        b. Windows: WordPad or NotePad should be free, standard text
           editors within the Start > Programs > Accessories folder.

        c. Mac OS X:
                    i. The included Applications > TextEdit can be used
                       only after the proper menu has been called:
                       Format > Make Plain Text
                                             L02 – page 19
BIOCHEMISTRY 660 / 712 – FALL 2006                                                   J.Y. SGRO


                         ii. Applications > Classes > BBEdit is an older
                             freeware program that offers many options and will
                             be used in this class. (Note: newer versions are
                             shareware).

       d. General Unix/Linux/MacOSX
Any file created with vi, pico, xedit, or a redirect command such as
cat > is created as a plain text files.

How to deal with line breaks or end-of-lines problems:

     A “hard-return” is coded within the text file. A “soft-return” is the apparent wrapping of a
     long text file to the next line, but in fact is a very long, single line. When saving from
     MSWord, one of the options is to save the file with Line Breaks that will in fact transform
     the soft- into hard-returns. For scripts this is not a good idea.

Hard-returns are coded as an operating system-specific
code. There are three options: Macintosh, Unix and DOS.
Macintosh refers to Macintosh OS Classic (through Mac OS
9.x), Unix is for Unix, Linux, and Mac OS X. DOS is for the
old Disk Operating System that has evolved into Windows.
With BBEdit, one can switch between the 3 hard-returns
with a single mouse option as seen on this image:

If you import a file from another system or the web, it may be a good idea to verify it’s end-of-
line / hard-return status if it does not work as expected or not at all within PyMol.

    3) Exercise scripts directory:

        For this exercise the script will be saved on the Desktop.

    4) If PyMol is running, Quit PyMol and restart it again, to have a fresh start. If you are
       starting here to learn about scripts, review how to find and start PyMol in exercise B.

    5) Use TextEdit (as detailed above) or BBEdit to create a new script that we shall call
       abc.pml. Save abc.pml on the desktop.

    Within abc.pml file type the following lines (comment lines which start with #. You can
    omit typing anything after the comment sign # for these exercises. However, placing
    comments within your script is a good practice for your future reference or for those with
    whom you share your scripts with, and can save hours of frustrations!)



    # this is my first script:
    load 2BIW.pdb2
    bg_color white
    hide lines
    show cartoon
                                              L02 – page 20
BIOCHEMISTRY 660 / 712 – FALL 2006                                                   J.Y. SGRO


    color red, ss h
    color cyan, ss s
    color yellow, not ss h and not ss s

    Note the use of the comma (,) after the name of the color.
       ss signifies secondary structure
       h signifies helix. (Note that the word “helix” would not be recognized.)
       s signifies sheet. (Note that the word “sheet” would not be recognized.)
       not h and not s are be the loops or turns.

    6) In your new PyMol session, set the default directory by typing the     cd Desktop
       command within the top line command. Then verify that the path is correct with the
       command pwd (present working directory):

        cd Desktop
        pwd

    7) Invoking a script: the @ command

             Rule: a script is invoked with the @
             command after its name within the line
             command: example: @abc.pml



              Type @abc.pml within the line command.

              Note: either the top (External GUI) or bottom (Internal GUI)
              line command can be used.



Activating the script will create the image as you could have done it by either typing each of the
script commands within the line command, or by using some of the mouse options as we have
done before. The advantage of scripts is that they are an easy way to create images in a
reproducible fashion, but most importantly, it can be an easy way to remember how the images
were created.

This image was created with the default orientation that the molecule takes when it is opened.
But very often, one important aspect of an image is the orientation of the molecule. PyMol
offers a very nice way to return to an existing orientation and build it within a script: the “Get
View” button, located at the top right of the “External GUI.”




                                                   L02 – page 21
BIOCHEMISTRY 660 / 712 – FALL 2006                                                       J.Y. SGRO




Rotate the molecule in an orientation you like
Click on Get View button
   The rotation matrix is shown on the PyMol text window, but is also placed within the
   clipboard.
Paste the matrix at the end of the abc.pml script
Add # in front of the load command to read: # load 2BIW.pdb2
Rotate the molecule again in another orientation or you can just type Reset instead to go
back to the default opening view.
Rerun the script by typing @abc.pml on the line command.

The PDB file will not be reloaded since we commented out the line with the load command (#).
The formatting and coloring of the protein will occur unnoticed since they are already in the
cartoon state colored by our chosen secondary structure colors. But the orientation of the
molecule will be restored.

The PyMol way: this is a nice way to work with PyMol. Simply create and modify a script as you
go, commenting out the loading of the PDB file(s) after the first run. It is a preferred way of working
with PyMol that leaves the legacy of a finished script with complex commands. Therefore you can
create your own repository of complex scripts, as a way to safeguard these commands for future
use and reference. In addition, you can run the script again and again as you build it, line by line.

                         =========================================

PyMol - Exercise H: Select command, parameters, scripting, and subsets.

        Understanding the content of this exercise can be beneficial to your PyMol skills!

The PyMol “select” command has the special property to create atom selections with names
appearing within the Names Panel. This is a nice feature, but some confusion can arise
depending on the words that are used. For example, in the abc.pml script we have just used
the command “color red, ss h” to colors red only the secondary structures (ss) that are
in helix (h) form. However the command “color red, ss helix” would not at this time
have any effect, and PyMol might give us an error message. The reason is that “helix” has no
meaning at this point. Interestingly this command CAN work if “helix” is defined first. “helix” can
be defined when an atom selection is created with the “select” command. Any subsequent
command can then contain the chosen word, and the commands will only apply to that atom

                                              L02 – page 22
BIOCHEMISTRY 660 / 712 – FALL 2006                                                                   J.Y. SGRO


selection, which can be a subset of a larger selection. When switching from Rasmol this can
be confusing, as the command “select helix” is a valid command on the Rasmol command line.

                 1) Defining by selection: an illustrated example

First let’s assume that you just ran the abc.pml script above on a fresh start of PyMol. If not, simply quit and
restart PyMol, then in the line-command area type cd desktop, press return and then type @abc.pml to be in
the same state as if you just had done the previous exercise. In the script the helices are made red. Here we will
make them green.

Within the line command type:                             PyMOL>color green, helix
                                                          Selector-Error: Invalid Selection Name.
color green, helix                                        helix<--

Note the error echoed within the PyMol text window

Within the line command type:            Two things will happen: a new name “(helix)” will appear
                                         within the Names Panel, and all the atoms participating in a
select helix, ss h                       helix structure will be selected within the Viewer (pink
                                         squares):




Within the line command type:            Note: this time it worked and the helices were colored green.
color green, helix

Note: The (helix) selection can be deleted with the command:
delete helix, however the name within the Names Panel only
disappears when the mouse is clicked within it. Alternatively
the seletion can be deleted with the “A” Actions menu “delete
selection.” The next menu allows to rename the selection.

Important lesson: the word “helix” here could have been any
other word such as “MyHelixSelection.” Using a “My” prefix
before some of the selections might help avoid confusing
words that are commands and words that are the names of
created selections. This can be important when writing or
deciphering other people’s scripts!                                              …any word can do…


                 2) PyMol Selectors: keywords to make atom selections

Reminder: PDB (and other) 3D coordinate files are structured with certain fields arranged in columns. PyMol
can detect and interpret some of these fields. For example, in the ATOM records, the “atom name column” or
“atom name field” is the column where the atoms are given a name, such as C, CA, CB, CG, or CD for example,
corresponding to the asymmetric carbon, the alpha-, beta-, gamma- and delta- carbons of an amino acid
respectively. Similarly, there will be columns with the name of amino acids, and clearly more than one line of
ATOM records make up for all the atoms of one amino acid. Finally, a chain and sequence number are also in
column.
                                                   L02 – page 23
BIOCHEMISTRY 660 / 712 – FALL 2006                                                                          J.Y. SGRO



               ATOM    3752   N      GLN   B   489     44.222    1.408    52.889   1.00   68.12             N
               ATOM    3753   CA     GLN   B   489     45.155    0.463    53.497   1.00   70.72             C
               ATOM    3754   C      GLN   B   489     45.787    1.050    54.759   1.00   71.44             C
               ATOM    3755   O      GLN   B   489     46.202    2.212    54.757   1.00   71.87             O
               ATOM    3756   CB     GLN   B   489     46.237    0.102    52.476   1.00   70.38             C
               ATOM    3757   CG     GLN   B   489     46.909   -1.223    52.738   1.00   72.85             C
               ATOM    3758   CD     GLN   B   489     47.621   -1.821    51.503   1.00   73.43             C
               ATOM    3759   OE1    GLN   B   489     47.815   -1.162    50.482   1.00   75.73             O
               ATOM    3760   NE2    GLN   B   489     48.014   -3.086    51.614   1.00   76.50             N


The complete list of selectors can be found on the PyMol manual
(http://pymol.sourceforge.net/html/index.html) or it’s echo as the wikipedia
version (http://www.pymolwiki.org/index.php/)

In summary, here are the most relevant selectors for working with macromolecules (proteins
and nucleic acids)

(Each definition below is followed by an example. Remember: as above, the word after the
word “select” is of your own creation. Adding “My” to the word may be a good reminder of that.)

 Matching Property Selector                                          Identifier and Example
                                           chemical-symbol-list
symbol                                     list of 1- or 2-letter chemical symols from the periodic table
                                           PyMol> select Mypolars , symbol o+n
                                           atom-name-list
name                                       list of up to 4-letter codes for atoms in proteins or nucleic acids
                                           PyMol> select Mycarbons , name ca+cb+cg+cd
                                           residue-name-list
resn                                       list of 3-letter codes for amino acids
                                           PyMol> select Myaas , resn asp+glu+asn+gln

                                           list of up to 2-letter codes for nucleic acids
                                           PyMol> select Mybases , resn a+g

                                           residue-identifier-list
                                           list of up to 4-digit residue numbers
                                           PyMol> select Myresidues , resi 1+2+20+8590
resi
                                           Residue-identifier-range
                                           PyMol> select MyNterm , resi 1-25
                                           chain-identifier-list
chain                                      list of single letter (rarely numbers) of the chain
                                           PyMol> select MyChain , chain a
                                           Secondary-structure-type
ss                                         list of single letters
                                           PyMol> select Myalphas , ss h+s+l+””

                 3) Putting it together: a fancier script

Lets make a last script to wrap it up. Create a script file called abc2.pml with BBEdit or other
means, and save it on the desktop.

Within abc2.pml type the following script. At this point you can omit typing the commented (#)
lines, but remember that placing comments within your scripts (annotation) can save (lots of)
time and remove headaches in the future…
                                                        L02 – page 24
BIOCHEMISTRY 660 / 712 – FALL 2006                               J.Y. SGRO



# my fancy script (assumes cd desktop for these exercises)
# first lets reset everything without need to quit PyMol
delete all
# set some parameters, even if some were done in previous
# white background:
bg_color white
# antialias: 1 for smooth images, 0 for jagged
set antialias = 1
# now reload the PDB file
load 2BIW.pdb2
# hide everything (all lines and so on)
hide everything
# show cartoon ribbon for the protein
show cartoon
# keep standard helix, strand and loops representations.
# other options would be: cartoon loop, cartoon rect,
# cartoon oval, and cartoon tube.
cartoon automatic
# make the helices with edges as in Molcript.
# 1 is on, 0 is off
set cartoon_fancy_helices=1
# color the inside of the helices in gray
set cartoon_highlight_color, gray
# color everything gray, and change other things later
color gray
# make selection of alpha helices and color it
select myhelix, ss h
color red, myhelix
# make selection of beta sheets and color it
select mybeta, ss s
color yellow, mybeta
# The PDB file contains a carotenoid ligand named 3ON
# it carrot when selecting it, show it as stick,
# and color it orange
select carrot, resn 3ON
show stick, carrot
color orange, carrot
# change the stick thickness from the default 0.25 to 0.40
set stick_radius = 0.40
# Inspection of the PDB file reveals there is also an iron
# select it and name it myiron, color it green and show it
# as a sphere. Then increase the sphere look quality.
select myiron, symbol FE
show sphere, myiron
color green, myiron
set sphere_quality = 8
# Select all full amino acids within 4 angstroms of the ligand
# and call this selection locals. Show them as a blue surface.
select locals, byres (chain B and carrot) around 4
show surface, locals
color blue, locals
# make the surface 15% transparent (85% opaque)
set transparency, 0.15
# Rotate about the Y axis to see the inside of the pocket
rotate y, 145, all
                                     L02 – page 25
BIOCHEMISTRY 660 / 712 – FALL 2006                                                   J.Y. SGRO


rotate z, -45, all
# zoom in on the ligand
zoom carrot
# make sure nothing is selected to avoid the little pink
# squares.
deselect
# end of this script – you could uncomment the next line to
# make a fancy image:
# ray


Within the PyMol line
command type @abc2.pml
to activate the script.

Your screen should be similar
to this image, which is the
ray-traced version.




                 4) Where to go from here ?

Writing a script may seem tedious at first, but once it’s written it can be saved, rerun,
upgraded, placed on the web, and exchanged with other people.

                   Phylosophy: Writing scripts is the PyMol way!

Where do I find the relevant variables such as sphere_quality?

The top menu cascade Setting > Edit All… opens a
window that lists all the variables and their current value.
The values can be changed interactively within this window,
or changed in the script as we have just done. This image
shows the current value for “sphere_quality” after running
the script: it has now the value 8, given by the script.




Learning by example: this is what we have done so far, at a slow pace. However there are
many web sites that offer PyMol help and scripts, often annotated with the # comments to
explain what is inside. Studying other’s scripts is a way to learn some fancy options. Here are
a few web sites to study at your own pace:
                                              L02 – page 26
BIOCHEMISTRY 660 / 712 – FALL 2006                                                                  J.Y. SGRO


• Creating a eye-catching figure with Pymol: (a must see!)
http://www.doe-mbi.ucla.edu/~sawaya/tutorials/Graphics/pymol.html

• Image gallery with corresponding PyMol scripts
http://www.chem.ucsb.edu/~molvisual/dna_biochem.html

• PyMol tips/scripts and tutorial:
http://www.rubor.de/bioinf/pymol_tips.html
http://www.rubor.de/bioinf/pymol_tutorial.html

• Introduction to Using PyMOL
http://pymol.sourceforge.net/carly/

• Visualizing Protein Structures - A Practical Introduction to PyMOL
http://www.ii.uib.no/~pal/teaching/mol305/files/pymol_intro.pdf

• Brief PyMOL tutorial:
http://www.mrc-lmb.cam.ac.uk/rlw/text/pymol_tutorial.htm

• Closed Configuration Of Dna Polymerase I
http://www.doe-mbi.ucla.edu/CHEM125/tutorial_1lv5.html

• PyMol & Movies
http://davapc1.bioch.dundee.ac.uk/teach/pymol/index.htm

• My PyMOL Script repository (for advanced use, including Python langage)
http://adelie.biochem.queensu.ca/~rlc/work/pymol/


         End of PyMol tutorial. Close or Quit all programs before leaving the classroom.

                         =========================================
                                           PyMol Q&A
                              (does not have to be completed in class)

    1) How can I work with PyMol on a very slow (laptop) computer?

The Display > Quality menu cascade allows you to adapt to the           Display
speed of you machine CPU depending on the quality of the image
displayed in the Viewer. This option MAY affect the result of ray-
traced images.

    2) Labels are created the same color as the atom. How can I change only the label color without changing
       the display of the atom or molecule?

You can change the label color with the following line command: set label_color, yellow, sele
• Yellow can be changed for other colors (can be chosen from the names within the C menu)
• Sele can be either the current selection, or can be switched the the name of the object you are working on.
• note the use of the comma (,) within the line command

    3) How can I make a new object?

The current selection (sele) as well as other subsets (e.g. (MyHelix)) are shown within parentheses and represent
subsets of the current PDB file we are working on (e.g. 2BIW2.pdb2). Any selection can be copied into an
independent object with the “A” Actions: create object menu cascade. This will create a new entry within the
“Names Panel” without the parentheses that becomes independent of the original PDB file.

For example, the ligand can be made into a separate entry:
                                                 L02 – page 27
BIOCHEMISTRY 660 / 712 – FALL 2006                                                                       J.Y. SGRO


        click on the ligand (becomes the current (sele))
        click on the A on the (sele) line and choose: create object (a new entry called obj01 appears within
        Names Panel, and an automatic zooming occurs)
        click on the S on the (obj01) line to change the object appearance, e.g. as stick
        If you make obj01 invisible by clickin on the obj01 icon, the ligand appears still here as it is also part of the
        2BIW.pdb2 file.

    4) Why can’t I make a surface for the ligand itself?

Surfaces are reserved for ATOM records only. Typically ligands are always HETATM records (but authors of the
PDB file may choose otherwise, hence always check within the file with a word processor.)

One way to circumvent this is to make the ligand into an independent object (see question above about creating
object), save it from PyMol into a text file, replace the ATOM word with HETATM and reopen the file with PyMol:

Assuming you have created obj01 as described above, use the menu cascade
File > Save Molecule….

Choose obj01 within the Save Molecule panel.

Choose a file name and save the file e.g. obj01.pdb. The file will be a PDB file
with only the object 3D data and contains HETATM and CONECT records.

Using a word processor open the PDB file and change replace “HETATM” with “ATOM “ (ATOM with 2 blank
space to respect the column positions!!!!!)
Delete the current obj01 with the menu cascade: Names Panel: obj01 > A >
delete object

Open the PDB file obj01.pdb with PyMol (menu cascade File> Open or use the
“load” line command)
A new obj01 now appears within the Names Panel, and the S > surface menu is
now working for this object!
                                                                                          ray-traced example

    5) How can I turn off the automatic zooming and clipping when I make a new object?

When we created object obj01 from (sele) above (see question about creating
objects), the default action of PyMol was to automatically zoom to the new object
(in that case the ligand) and reduce the clipping planes. That can be annoying in
some cases, but could always be reversed with the line command “reset” or with
the menu Display > Clip > None or with the Display > Zoom menu options.

To turn off this option is turned off by un-checking the menu item:
Setting > Auto-Zoom New Objects



    6) Can I superimpose in 3D two similar structures?

PyMol will have no problem aligning 2 similar structures. PyMol firsts creates a sequence alignment the then tries
to align the structures accordingly. If 2 proteins are named struct1 and struct2 within the Names Panel, the simple
line command will align them: align struct1, struct2 (note the use of the comma after struct1)

For more complex alignment questions, see the following web site:
http://adelie.biochem.queensu.ca/~rlc/work/teaching/BCHM823/pymol/alignment/



    7) Where is the HELP command?

                                                     L02 – page 28
BIOCHEMISTRY 660 / 712 – FALL 2006                                                                 J.Y. SGRO


Some on-line web content can be called from the Help menu in PyMol.

PyMol also offers an internal summary of all the command derived from the manual. These are called from the
line command help xxx where xxx is the name of the command, e.g. help align or help color.

    8) I have an NMR file called 1NYZ with 20 structures… How can I see or work on a specific state?

If you open the PDB file with a word processor, you can see that each structural state starts with the MODEL
keyword and is separated from the next with the ENDMDL keyword. PyMol will read all molecule states, in this
example 20. This is also echoed within the text panel:
e.g. ObjectMolReadPDBStr: read MODEL 17

When the file is opened, a single name (e.g. 1NYZ) will be
shown within the Names Panel and a single molecule (state)
is shown on the Viewer. At this point all actions will apply to
all structures at the same time, e.g. displaying as stick model
Names Panel : 1NYZ > S > stick
If you click on the VCR triangular play button at the very
bottom right, you will see an animation showing each state
one after the other. You can also save this animation as a
movie with the File > Save Movie As… menu option.

The following line-command will separate all the structure
and make each one of them a separate object assuming the
name of the object is 1NYZ: split_states 1NYZ

When the command is given, 20 new objects are created,
labeled from 1NYZ_0001 to 1NYZ_0020. These can be
worked on individually with the help of the ASHLC menus
within the Names Panel or made invisible by simply clicking
on its name in the Names Panel.




    9) How can I keep a log of the line commands I type?

All commands are echoed on the Text panel together with any additional text
triggered by the command. While it is possible to use Copy/Paste from this panel, it
is much more convenient to have a separate file logging all the typed commands
only. Furthermore, this log file is de facto a script!

The logging can start after the File > Log… command is requested and a file name
is given, e.g. test.log. The file can be saved anywhere on the hard drive, for
example the desktop. The menus allow to close the current log, resume logging or
append to any previously created plain text file.

                                                                                       cd desktop
You can try to create a log file while typing the commands on the right hand side      load 2BIW.pdb2
                                                                                       select hetatm
on the PyMol line command.
                                                                                       show stick
                                                                                       hide stick
You can then quit and re-open PyMol and run this test.log file with the following      show stick, sele
command: cd desktop; @test.log                                                         color yellow, sele

Note: if you want to run it from the File > Run… menu, you may need to rename the file with a .pml file extention
e.g. test.log.pml otherwise the file cannot be opened.

Practical note: previously typed command on the line-command can be recalled by using the “up-arrow” key.

                                                    L02 – page 29
BIOCHEMISTRY 660 / 712 – FALL 2006                                                                    J.Y. SGRO



    10) When I make a cartoon helix, part of the helix color bleeds on to the turns, how can I fix that?

Turning the variable cartoon_discrete_colors on makes the helix color ends abruptly at the end of the helix. The
default value is off. The change can be done manully with the menu cascade Setting > Edit All… or can be given
as a typed command: set cartoon_discrete_colors = on (space on either side of the = sign is optional)

Setting
Edit All…




                                        set cartoon_discrete_colors = off         set cartoon_discrete_colors = on


    11) I want to build a model helix from a sequence, how can I do that?

PyMol offers modeling options for small molecules and proteins. For example you can build an alpha helix from
scratch with a specific sequence. The “builder” interface to accomplish this is different depending on the operating
system. On Windows and X11 versions of PyMol, the “External GUI” has an extra set of buttons to build by
clicking on the various components to be assembled such as amino acids. The MacPyMol version has the same
capability but instead of clicking icons menu items are chosen.

Windows builder interface:
Small Chemical molecules builder panel:




Protein builder panel:




MacPyMol builder interface is via the Build menus:
Chemical molecules builder panel:               Protein builder panel (split in 2):




                                                                                       continued…>

Steps to build a model helix:

                                                    L02 – page 30
BIOCHEMISTRY 660 / 712 – FALL 2006                                                                  J.Y. SGRO



            a.   (Windows: click on “Builder” button at right and then on “Protein button at left.)
            b.   Choose the helix form (bottom of menu Build>Residue. (Windows: click on “Next”)
            c.   Select the sequence you want to build from the Build (Windows: clicking residue name)
            d.   The model will be built withint the Viewer.
            e.   Save the molecule with the File > Save Molecule menu. The name of the molecule will be the
                 name of the first amino acid used e.g. ala.pdb

                        The alpha helix is
                        created as a “perfect”
                        model and contains all
                        hydrogen atoms.


    12) How can I mutate one side-chain from an existing structure?

The menu cascade Wizard > Mutagenesis opens a new panel below the Names Panel and above the mouse
control reminder. Directions will be prompted with text overlaid on the Viewer:
“Pick a residue” and “Select a conformational state, or pick a new residue…”

Steps to mutate one amino acid on a protein structure:

         a. Open from the menu Wizard > Mutagenesis


         b. Click on the residue you wish to mutate
         c. Select a conformational state in the new mutageneis panel menu (bottom
            right)
            (options are backbone dependent or independent)
         d. Click on the No Mutation button and select a new amino acid (e.g. ARG)
         e. Click Apply
         f. Repeat process for mutating more residues
         g. Click Done when finished with the Mutagenesis Wizard.

    13) I’d like to try various way of orienting, drawing and coloring a structure. How can I compare them?

While PyMol cannot show all the versions at the same time, the Scene menu has very nice feature to save the
structure in various states of representations and toggle between them. The transition from one scene to the next
creates also a beautiful screen animation.

Simple example with file 2BIW.pdb2:

            a. Create a first version of a representation: show as cartoon (S > cartoon) and color by secondary
               structure (C > by ss > pick-a-color-scheme). Now orient the molecule with the mouse in a way
               you like.
            b. Store this view in the F1 keyboard key with the menu cascade Scene > Store > F1
            c. Create a second presentation for example change the cartoon color (C > spectrum) and rotate
               the molecule in a different orientation. For a more stunning effect you can zoom in.
            d. Store this new view into the F2 key: Scene > Store > F2
            e. Now you can recall the previous scene with the menu cascade Scene > Recall > F1 and you
               should witness a beautiful animation and color change while the scene is changing.

    14) How can I rotate 2 molecules relative to each other?

There are 2 ways to accomplish this task: with the mouse or with line command.

Mouse method:



                                                   L02 – page 31
BIOCHEMISTRY 660 / 712 – FALL 2006                                                                    J.Y. SGRO


            a. Switch to 3-botton mouse editing with the Mouse menu
               The table at bottom right summarizes the possible
               movements. The mouse movement ending with “O” are
               related to object rotations: Shift+Left button = RotO (rotate
               object). Shift+Middle button= MovO (move object), Shift+
               Right button=MvOZ (translate the object along the Z axis).
            b. Click on the object (molecule) you want to rotate or
               translate.

            c.   Apply the proper mouse/keystroke combination for rotation, translation, or moving along Z
                 (toward or away from you)

Line command method: learning by example.

The line command method is shown here by an           load 1FJ1.pdb
example from the PyMol author.                         
                                                      # split PDB file
                                                       
1FJ1.pdb contains an antigenic fragment and its       create anti=(chain F)
bound antibody in a single PDB file.                  create fab=(chain A,B)
                                                       
Once the PDB file is read in with the load            # delete original object
command, the relevant protein chains are copied       delete 1FJ1
into independent objects and given a name              
                                                      # color objects
(anti and fab.)                                       color green,fab
                                                      color pink,anti
Some selection and coloring is done to help            
visualize.                                            # color interface
                                                      select inter = (byres ((fab within 5 of anti)\
inter is the name given to the interaction area.         or (anti within 5 of fab)))
                                                       
byres helps select complete rather than partial       color yellow,inter
amino acids.                                           
When a line is long in a script, the charater \       # splay apart
indicates that the command continues on the           orient
next line.                                            origin fab
                                                      rotate y,60,fab
                                                      origin anti
The command orient orients the molecule               rotate y,-60, anti
along the XYZ axes.                                    
The command origin places the center of               # zoom interface region
rotation on the deginated object, which is then       zoom inter
rotated around the y axis with the command            show sph,inter
                                                      disable inter
rotate.

Provided the PDB file 1FJ1.pdb (http://www.rcsb.org) is available, this script called split.pml can be run from the
line command with @split.pml or called from the File>Run… menu cascade IF the filename extension is .pml.

    15) How do I make an electrostatic potential surface map?

The most accurate potential maps can be calculated by external software (Grasp, Delphi, APBS, MEAD) and
displayed in PyMol. Search the web or the Pymol usergroup archive for details on using external software to
create a map http://sourceforge.net/mailarchive/forum.php?forum_id=60 or see this posting
from PyMol author: http://imsb.au.dk/pipermail/o-info/2004-December/007601.htm




                                                    L02 – page 32
BIOCHEMISTRY 660 / 712 – FALL 2006                                                                     J.Y. SGRO


PyMol offers an approximate map nicknamed "charge-
smoothed" surface representing approximate charge
distribution on the protein surface. Assuming that you have
opened PDB file 2BIW.pdb2, the potential surface is
calculated and displayed with the following menu cascade
on the Names Panel:

2BIW.pdb2 > A > generate > vacuum electrostatics > protein contact potential (local)
The process creates 3 new entries within the
Names Panel: 2BIW.pdb2_e_chg,
2BIW.pdb2_e_map, and 2BIW.pdb2_e_pot
(2BIW.pdb2 is subsequently abbreviated X
below.)

X_e_chg is an object containing the surface,
colored red/white/blue and is an approximate
map.
X_e_map is usually not shown (click on name
to show) and displays the volume boundaries
as e.g. a big cube.

X_e_pot is the object representing the color
ramp and value at the bottom of the display.

    a. adjusting the value range and color strength:

If two proteins are present, the process has to be applied to each individual protein, and the same _e_ objects are
created for each one. The values within the X_e_pot color ramp may be different for each protein. Therefore it is
useful to know how to change the value within. Smaller numbers will increase the blue and red strength and
contrast within the blend of white surface, while larger numbers will dim the colors. To change the color range
within the ramp: (example): ramp_new 2BIW.pdb2_e_pot , 2BIW.pdb2_e_map, [-100,0,100]

    b. Adjusting the display colors:

The default colors are red/white/blue, hence the above command could also be rewritten to change ramp values
and specify colors:

ramp_new 2BIW.pdb2_e_pot , 2BIW.pdb2_e_map, [-100,0,100] , [ red, white, blue]
or
ramp_new 2BIW.pdb2_e_pot , 2BIW.pdb2_e_map, [-100,0,100] , [ [1,0,0], [1,1,1] , [0,0,1] ]

where [ [1,0,0], [1,1,1] , [0,0,1] ] represents the [R, G, B] (red/green/blue channels) values for each of the 3
displayed colors. To change the displayed color, simply change the definition of the colors. For example, the
values [ [1,1,0], [1,1,1] , [0,1,1] ] would create a ramp as yellow/white/cyan.

Note: an alphabetical list of all PyMol-defined color names can be found under the top menu “Setting > Colors…”
Within that window the colors can also be edited.
The C menu within the Names Panel offers a list of color names grouped by tint and can serve as a preview.

    c.   Default display:

Note that when the calculation first takes place, the surface is shown but the original PDB file is hidden. In the
case of 2BIW it means that the carotenoïd becomes invisible. To restore it, simply click on button 2BIW.pdb2
within the Names Panel: this will restore the display of the protein and ligand as lines or sticks. Since the protein
is under the surface it will remain invisible (but some odd side chains might stick out of the surfaces.) To make
more complex images it may be useful to create a new object containing the ligand alone (see questions above:
How can I make a new object? and Why can’t I make a surface for the ligand itself?)
                                                     L02 – page 33
BIOCHEMISTRY 660 / 712 – FALL 2006                                                                J.Y. SGRO



    16) How can I display hydrogen bonds?

See also “Polar Contacts” within the following web page:
http://www.pymolwiki.org/index.php/Displaying_Biochemical_Properties

“Polar Contacts” can be displayed with the menu
A > find > polar contacts and selecting from one of the
many submenus.

“within selection” and “to other atoms in object” are
particularly useful.

Example to display the hydrogen bonds within the alpha
helix consisting of residues 94 to 105 in 2BIW.pdb2:

First create object helix-1 containing the helix residues
with the line command:

select helix-1, resi 94-105

Then within the Names panel follow the menu cascade:

helix-1 > A > find > polar contacts > within selection

A new objects 2BIW.pdb2_polar_conts will be created. The yellow default color for the dashed line is easily
changed with the C menu for this object. For example a darker color can be chosen for display against a white
background.

Note: choosing to other atoms in object would show additional hydrogen bonds of the helix side-chains to other
parts of the protein.

Note: for more complex issues regarding hydrogen bonds or adding hydrogen atoms refer to the web link above.

    17) END of Q&A.

                                =========================================




                                                     L02 – page 34
BIOCHEMISTRY 660 / 712 – FALL 2006                                                   J.Y. SGRO



Reply to a student’s Question: Is there a way in pymol to make a quick time
movie that rotates in the x, y, and z direction at the same time?

For a rotation within PyMol it’s easy, and the command is:

mdo 1: turn y,5; turn x,5; turn z,5;

for 5 degrees rotations on each angle

I agree with you that the PyMol movie making is not as straight forward as it sounds for more
elaborate movies.

First, we shall assume that you are using a PDB file with one "state." In other words you are
not using an NMR file that contains multiple structures (refer to the addendum I gave in class
if that confuses you, relative to NMR file 1NYZ.pdb.)

For movies in PyMol, the first thing to understand is that you have to specify the number of
frames at the begining of the movie commands. THis is done with mset, but mset is defined
in different ways depending on what you are doing. Assuming you are using a one state PDB
file, you need to state how many frames the movie is going to be:

mset 1 x30            # creates a 30 frame movie consisting of state 1 played 30 times.

you can review more mset options on the following link, but I think that you should not need
any of the other options for your rotations.

http://pymol.sourceforge.net/newman/user/S0300movies.html

After some research on the net here are some solutions I have found to your question about
rotating about the 3 axes at the same time. The answers were not as easy to find and I am
not sure if they completely answser what you want.

1) the easiest:
===========

In some way I assume that you want to make a "nice" rotation that is not as boring as the
simple Y rotation. In this case the command movie.nutate would be helpful and most likely
what you are looking for. With a small angle it resembles the default movement "lemniscate"
in VMD movie maker. Here is an example: (# is the comment symbol and everything is ignore
by PyMol after it.)

mclear      # clears out previous movies you might have tried
mset 1 x72  # uses state 1 for a 72 frames movie
movie.nutate 1,72,360    # starts with frame 1 until frame 72
                         # for a 360 rotation around all axes.

Of course the movie looks better with more frames and more angles:


                                             L02 – page 35
BIOCHEMISTRY 660 / 712 – FALL 2006                                                  J.Y. SGRO


mclear
mset 1 x360
movie.nutate 1,360,360

movie.nutate belongs to the built-in PyMol Python script movie.py and has more options
as I have found at the following site:
http://www.koders.com/python/fid54F75215FAB3B81AE8E0824786C2FD828C60845C.aspx

def nutate(first,last,angle=30,phase=0,loop=1,shift=math.pi/2.0,factor=0.01)

so you can see that the first number is the first frame that will be affected. You could decide to
start moving only after frame 10 for example. The second number is the last frame that is
going to be moving with the rotations. The third number is the rotation desired. If you use e.g.
20 instead of 360 you will have the "Leminscate" effect seen in VMD. The next numbers can
be played with to test their effect(s) but are not mandatory to the command.

NOTE: the use of movie.nutate was suggested to a similar question by a user to the
PyMol author Warren Delano. See:
http://sourceforge.net/mailarchive/message.php?msg_id=2078718

2) Using cmd.mdo
==============

This one is inspired from the following answer on the sourceforge archive:
http://sourceforge.net/mailarchive/message.php?msg_id=9881931

mclear
mset 1 x360
for a in range(1,360): cmd.mdo(a,"rotate x,5;rotate y,5;rotate z,5")
You will note that the rotation is not as smooth as the movie.nutate option, even with a
rotation of 1 rather than 5.

3) Related question: rotating around Y and then X....
======================================

Answer from link:
http://sourceforge.net/mailarchive/message.php?msg_id=2371062

 mset 1 x360
 movie.roll 1,180,1,axis=y
 movie.roll 181,360,1,axis=x

Frames 1 to 180 are for Y rotation and frames 181 to 360 are for X rotation. You could
decompose the frames in such a way to add a Z rotation as well:

 mset 1 x360
 movie.roll 1,120,1,axis=y
 movie.roll 121,240,1,axis=x
 movie.roll 241,360,1,axis=z

                                          L02 – page 36
BIOCHEMISTRY 660 / 712 – FALL 2006                                                 J.Y. SGRO


However there is another command that can do all that in one line: movie.tdroll:

AUTHOR
Byron DeLaBarre
USAGE
movie.tdroll(rangx,rangey,rangez,skip=1,mset=0)
rangex/y/z = rotation range on respective axis
enter 0 for no rotation.
skip is angle increment in each frame
Use skip to reduce final movie size or to speed up rotation.                EXAMPLE
movie.tdroll 360,360,360,5

as shown on link:
http://sourceforge.net/mailarchive/message.php?msg_id=1534644
 movie.tdroll ("Three-Dimensional roll") will allow you to use make a quick   movie
of rotation along multiple axes of whatever is showing on the pymol   screen. You
can speed it up or slow it down by changing the "skip" value.   Play with setting
different axes to 0 until you achieve what you want. I     like movie.tdroll
180,180,0,5 for a cool display of the molecule.

4) Saving your movie:
=================

If you are on a Mac, the menu File > Save Movie > As QuickTime should work with all the
examples above.

If you are on a PC the files will be saved as single PNG files that need to be assembled, e.g.
with QuickTimePro on a PC.

You can also export the individual frames with the line-command mpng NAME which would
save frames NAME0001.png, NAME0002.png etc until the last frame.

I hope this will get you to what you wanted to do....As you can see, a simple question can
lead to a very long answer....

Jean-Yves

P.S. I almost forgot.... There is a separate Python module that can be downloaded and then
invoked within PyMol. This module is not part of PyMol and needs to be downloaded, and
then activated within PyMol before the new commands are available

You can see an example of this at the following link:
http://sourceforge.net/mailarchive/forum.php?thread_id=1204166&forum_id=60

However the download link is not working, but I found the following link that contains the
Python script, but it is INCLUDED within the following archive at the bottom of the archive.

http://sourceforge.net/mailarchive/message.php?msg_id=4272454

The assumed name of the script is movie.py, however it is a different movie.py python script
than the one I mentioned above in the movie.nutate paragraph.
                                         L02 – page 37
BIOCHEMISTRY 660 / 712 – FALL 2006                                              J.Y. SGRO


This link also contains an example script on the usage of this. THe command "run movie.py"
activates the script which is simply kept in a text file.

Some of the meaning of the commands are at:
http://www.rubor.de/bioinf/pymol_movie.html
(but the download link does not work, see above).

NOTE: movie.py is not part of the standard PyMol installation, and any script making use of
movie.py commands would only work movie.py is available on the PyMol that is running.

                               =========================================




                                              L02 – page 38

								
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